Cytoplasmic and Nuclear Fractionation

RAW264.7 cells were seeded at a density of 1 × 10⁶ cells/ml on 100 mm cell culture dishes for 24 h in a 5% CO₂ incubator. Cells were pre-treated with CHE for 1 h and then treated with 1 µg/ml LPS for 30 min. Cells were collected after incubation and washed twice with cold PBS. Separation of cytoplasmic and nuclear proteins was performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to the manufacturer’s protocol. Separated proteins were then visualized with a western blot assay.